Arabidopsis calmodulin-like protein CML36 is a calcium
(Ca
2
ⴙ
) sensor that interacts with the plasma membrane
Ca
2
ⴙ
-ATPase isoform ACA8 and stimulates its activity
Received for publication, March 23, 2017, and in revised form, July 7, 2017 Published, Papers in Press, July 18, 2017, DOI 10.1074/jbc.M117.787796
Alessandra Astegno‡1, Maria Cristina Bonza§, Rosario Vallone‡, Valentina La Verde‡, Mariapina D’Onofrio‡, Laura Luoni§, Barbara Molesini‡, and Paola Dominici‡
From the‡Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy and the§Department of Biosciences, University of Milano, Via Celoria 26, 20133 Milano, Italy
Edited by Joseph Jez
Calmodulin-like (CML) proteins are major EF-hand– con-taining, calcium (Ca2ⴙ)– binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2ⴙsensors and identification of novel target proteins of CMLs deserve spe-cial attention. To this end, we recombinantly produced and bio-chemically characterized CML36 from Arabidopsis thaliana. We analyzed Ca2ⴙ and Mg2ⴙ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2ⴙ/Mg2ⴙmixed binding sites and two low-affinity Ca2ⴙ-specific sites. Binding of Ca2ⴙinduced an increase in the␣-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2ⴙor Mg2ⴙ
, sta-bilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro bind-ing and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma mem-brane Ca2ⴙ-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflores-cences. Collectively, our results support a role for CML36 as a Ca2ⴙsensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modula-tion of plant-autoinhibited Ca2ⴙpumps.
Calcium (Ca2⫹) is a crucial second messenger in plants,
where it couples the perception of endogenous and environ-mental signals to plant responses (1– 4). Ca2⫹signals are
trans-mitted by stimulus-specific cytosolic Ca2⫹ elevations that result from the concerted action of both Ca2⫹influx (channels) and Ca2⫹efflux (pumps and carriers) systems, which
tempo-rally shape and spatially define Ca2⫹ dynamics, commonly
referred to as the “Ca2⫹signature” (3– 6). These signatures are
believed to encode information from primary stimuli and, therefore, to contribute to the stimulus specificity of the biolog-ical response. Nonetheless, another layer of specificity in Ca2⫹
signaling is achieved via the function of Ca2⫹sensors, which detect alterations in intracellular free Ca2⫹ concentration
decoding specific Ca2⫹signatures into downstream physiolog-ical responses (7). Most Ca2⫹sensors harbor the EF-hand motif, a helix-loop-helix structure binding one Ca2⫹ion. The EF-hand
binds Ca2⫹ through a pentagonal bipyramidal arrangement, and the chelating residues are notated either based on their linear positions (1, 3, 5, 7, 9, and 12) or on their tertiary coordi-nation geometry (X, Y, Z,⫺Y, ⫺X, and ⫺Z) (8). In the canonical EF-hand, positions 1 (X), 3 (Y), 5 (Z), and 12 (⫺Z) are side-chain oxygen ligands, 7 (⫺Y) is a backbone carbonyl ligand, and 9 (⫺X) is a water ligand hydrogen-bonded to one loop residue (8). Ca2⫹binding modifies the conformation of EF-hand proteins
resulting in changes in enzymatic activity of the Ca2⫹sensor itself or allowing it to interact with and alter the target protein activity (9). Besides calmodulin (CaM),2which has been
well-conserved during evolution, plants contain a unique family (50 members in the model plant Arabidopsis thaliana) of CaM-like proteins (CMLs) that greatly differ from canonical CaMs in sequence (they share at least 16% overall amino acid identity with CaM2 of Arabidopsis), length (some CMLs possess N- or C-terminal extensions), and number of EF-hands (from 1 to 6) (10 –12). Functional analyses of various plant CMLs have pro-vided clear indications for the key roles of these proteins in plant development and abiotic and biotic stress responses, as overviewed in several recent reviews (12–15). Altogether, these studies do not support a functional redundancy hypothesis for CMLs, which could be key positive and/or negative regulator components in the coordination of plant responses to different environmental stimuli (15). However, to date only a few of these proteins have been clearly shown to function as Ca2⫹sensors by
describing their Ca2⫹-binding (e.g. dissociation constants,
stoi-chiometry, ligand specificity) and structural properties and by
This study was supported in part by Grant FUR2014 from the University of Verona (to A. A. and P. D.). The authors declare that they have no conflicts of interest with the contents of this article.
This article containssupplemental Figs. S1 and S2 and “Methods.”
1To whom correspondence should be addressed: Dept. of Biotechnology,
University of Verona, Strada Le Grazie 15, Verona (VR), Italy. Tel.: 39-045-8027955; Fax: 39-045-8027929; E-mail: alessandra.astegno@univr.it.
2The abbreviations used are: CaM, calmodulin; CML, CaM-like protein; ACA8,
Ca2⫹-ATPase isoform 8; CML36 f.l., full-length CML36; PM, plasma
mem-brane; ANS, 1-anilino-8-naphthalene sulfonate; SEC, size-exclusion chro-matography; ITC, isothermal titration calorimetry; HSQC, heteronuclear single-quantum coherence; DSS, 2,2-dimethyl-2-silapentanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
J. Biol. Chem. (2017) 292(36) 15049 –15061
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identifying their biological targets. Thus, a detailed character-ization of CMLs at the biochemical and structural levels com-bined with the identification of distinct downstream targets is essential in determining the high level of specificity that allows the plant cell to transduce diverse stimuli into proper physio-logical responses.
In the present study, we characterized CML36 from A.
thali-ana.In particular, using different complementary biophysical and biochemical methods, we analyzed whether CML36 pos-sesses the structural and binding properties of Ca2⫹sensor pro-teins. We found that CML36 behaves as a Ca2⫹sensor, in that it
experiences Ca2⫹-dependent changes in secondary and tertiary structure and in the exposure of hydrophobic regions. More-over, we identified a biological target, providing evidence that CML36 interacts directly with Arabidopsis Ca2⫹-ATPase iso-form 8 (ACA8), a type IIB Ca2⫹pump localized at the plasma membrane (PM) that contributes to the overall Ca2⫹
homoeo-stasis and to the control of intracellular Ca2⫹ signaling by actively extruding Ca2⫹from the cytosol into the apoplast (16 –
18). We identified the specific CML36-binding site at the N terminus of ACA8 and demonstrated that the interaction between ACA8 and CML36 promotes the pump Ca2⫹
-depen-dent hydrolytic activity in vitro. Collectively, our findings allowed us to classify CML36 as a Ca2⫹sensor, uncovering a possible involvement of CML36 in the modulation of the plant autoinhibited Ca2⫹pump.
Results
CML36 is a Ca2ⴙsensor
Recombinant production of full-length CML36 (CML36 f.l.) resulted in a highly unstable protein (with a high propensity to aggregate and precipitate) at the concentrations required for efficient protein-based biophysical and structural analyses. CML36 contains an extended N-terminal region (residues 1– 60) and a Ca2⫹-binding region (residues 61–209) with four EF-hand motifs. The extended N-terminal region was found to be remarkably intrinsically disordered using the GeneSilico MetaDisorder Service (19), and therefore a truncated variant of CML36 was created by deleting the first 60 residues. The result-ing mutant (residues 61–209, named CML36-C) was very stable
even at high concentrations and was therefore used for all bio-physical and structural analyses.
Typical features of Ca2⫹sensors include Ca2⫹-induced con-formational changes that often expose hydrophobic surfaces necessary for target recognition. Thus, we examined Ca2⫹ -de-pendent conformational changes and surface hydrophobicity of recombinant CML36-C by using an array of biophysical and structural techniques such as nuclear magnetic resonance (NMR), circular dichroism (CD), 1-anilino-8-naphthalene sul-fonate (ANS) fluorescence spectroscopy, and size-exclusion chromatography (SEC). Moreover, because of the high concen-tration of free Mg2⫹in plant cells (0.5–2 mM) (20, 21) we also
analyzed the behavior of CML36-C upon the addition of Mg2⫹, to determine the specificity of CML36 in discriminating cyto-solic Ca2⫹signals against a⬃102–104-fold excess of the chem-ically similar divalent cation Mg2⫹. Mg2⫹ binding to
EF-hands is physiologically important; distinct roles have been proposed for the binding of this ion (8, 22), such as a structural role, by conferring structural stability to an otherwise poorly defined molten globule apo-state, or roles in the Ca2⫹ -depen-dent regulation of cellular processes by modulating the affinity of EF-hands for Ca2⫹.
NMR spectroscopy was used to investigate the structural rearrangements of CML36-C upon the addition of metal ions. The signals in a two-dimensional1H-15N heteronuclear single-quantum coherence (HSQC) NMR spectrum belong to the HN groups of the protein. The frequency of HN signals (chemical shift) correlates with the chemical environment of the observed nuclei and is influenced by both local and global structure.
The1H-15N HSQC spectrum of apoCML36-C is
character-ized by the presence of fewer peaks than expected, severe line broadening, and poor chemical shift dispersion, especially in the proton dimension (Fig. 1A). These spectral features suggest that apoCML36-C undergoes conformational fluctuations typ-ical of a molten globule state, similar to other EF-hand proteins in their apoforms (23–25). A change in the HSQC spectrum of CML36-C was observed upon the addition of a saturating con-centration of Mg2⫹(Fig. 1B). The spectrum is characterized by
more uniform peak intensities and an increased chemical shift dispersion, indicating that Mg2⫹-bound CML36-C adopts a
Figure 1. Two-dimensional1H-15N HSQC NMR spectra of15N-CML36-C. The spectra were recorded at 600 MHz and 25 °C in the presence of 5 m
MEGTA (A),
5 mMMgCl2(B), and 5 mMCaCl2(C). All samples were at a protein concentration of 500Min 20 mMTris-HCl, 1 mMDTT, pH 7.5.
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more stable tertiary structure compared with the apoprotein. However, the number of peaks in the Mg2⫹-bound form
remained much lower than expected considering the non-pro-line residues in the amino acid sequence of CML36-C (150 res-idues), and many peaks in the central area of the HSQC spec-trum still appeared rather broad, meaning that some portions of the Mg2⫹-bound protein may not be structured or may still possess a high degree of flexibility. Remarkably, the addition of saturating Ca2⫹concentrations to the apoCML36-C resulted in a more dramatic change in the CML36-C HSQC spectrum, where⬃120 peaks with good chemical shift dispersion became visible (Fig. 1C), indicating that the addition of Ca2⫹promotes a global structural rearrangement throughout the protein. By comparing the position of the peaks in the two metal-bound forms, it was evident that Ca2⫹ induced conformational changes in CML36-C distinct and separate from those induced by Mg2⫹, which is in line with a putative Ca2⫹sensor function of the protein.
The far- and near-UV CD analyses were consistent with the interpretation of the apoCML36-C as a molten globule state, as in apoCML36-C the secondary structure is conserved to a large extent, whereas the tertiary structure is highly fluctuating.
Pan-els Aand B of Fig. 2 show the far- and near-UV regions of the CD spectra of apoCML36-C and Ca2⫹- or Mg2⫹-bound
CML36-C, respectively. As evident in Fig. 2A, the far-UV CD spectra for the apoform and both holoforms of CML36-C show two negative bands with minima at 208 and 222 nm, character-istic of proteins with a high␣-helical content. However, the reduced 222/208 ratio observed in apoCML36-C (0.67 for apoCML36-C versus 0.83 and 0.82 for Ca2⫹-CML36-C and
Mg2⫹-CML36-C, respectively) suggests the presence of some regions of high flexibility or random coil structures.
Despite the absence of Trp residues, CML36-C has two Tyr and eight Phe residues, and the corresponding bands are well-resolved in the near-UV CD spectra (Fig. 2B). Signals in the 275–290-nm range can be associated with the microenviron-ment of Tyr residues, whereas the bands observed in the 250 – 270-nm region, with minima at 262 and 268 nm, are ascribable to Phe residues. The switch of CML36-C from the holo- to the apoform produces considerable changes in this part of the CD spectrum (Fig. 2B), suggesting a partial collapse of the tertiary structure.
A common feature of Ca2⫹sensors is the increase in
surface-exposed hydrophobicity upon Ca2⫹binding, which we inves-tigated by the use of the fluorescent probe ANS. This dye is a hydrophobic compound that shows a blue-shift in the maxi-mum emission and a considerable increase in fluorescence upon binding to hydrophobic regions of proteins. In the pres-ence of apoCML36-C, we detected a⬃7-nm blue-shift and a modest increase in the emission of ANS in comparison with ANS alone (Fig. 2C), suggesting that apoCML36-C possesses exposed hydrophobic portions. The addition of Mg2⫹did not cause further changes to the spectrum, whereas upon the addi-tion of Ca2⫹ a further blue-shift (⬃12 nm) and a 1.6-fold
increase in fluorescence emission was observed, indicating that CML36-C undergoes a Ca2⫹-dependent exposure of
hydro-phobic surfaces.
Analytical SEC, together with pulsed-field gradient diffusion NMR studies, was conducted to examine the hydrodynamic radius (Rh) of CML36-C in the apo-state and the Mg2⫹-bound Figure 2. Ca2ⴙsensor properties of CML36-C. A and B, far-UV CD spectra of 10MCML36-C (A) and near-UV CD spectra of 90MCML36-C (B) in the presence
of 5 mMEGTA (solid line), 5 mMMgCl2(dashed line), and 5 mMCaCl2(dotted line). C, ANS-fluorescence of CML36-C in the presence of 5 mMEGTA (solid line), 5 mM
MgCl2(dashed line), and 5 mMCaCl2(dotted line). The spectrum of ANS alone (dash-dotted line) is also shown. D, SDS-PAGE mobility shift of CML36-C in the
presence of 5 mMCaCl2or 5 mMEGTA. Lane M is a molecular mass marker. E, native PAGE mobility shift of CML36-C in the presence of 5 mMCaCl2or 5 mMEGTA. at UNIVERSITA' DI VERONA on March 10, 2018
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and Ca2⫹-bound states (Table 1). Notably, in all of these states,
the obtained Rhresulted in an apparent molecular mass over-estimation, which has been observed in various Ca2⫹sensor proteins (26 –28) and is related to their extended conformation. Moreover, the binding of Ca2⫹, and to a less extent of Mg2⫹, to CML36-C determined a decrease in Rh, as found elsewhere for CaMs (26, 27, 29).
Altogether, these structural studies strongly suggest that CML36-C functions as Ca2⫹ sensor, indicating that
apo-CML36-C is in a dynamic molten globule form that undergoes a large conformational change upon binding Ca2⫹. Moreover, CML36-C displays a higher electrophoretic mobility in the presence of Ca2⫹both in denaturing and native conditions (Fig. 2, D and E), which is a well-documented phenomenon for CaM proteins (29 –32).
CML36 contains four Ca2ⴙ-binding sites
Isothermal titration calorimetry (ITC) was used to monitor Ca2⫹and Mg2⫹binding to CML36-C, which allows the deter-mination of accurate values of apparent dissociation constants (Kd) for multiple binding sites and related changes in enthalpy (⌬H). Representative ITC isotherms are shown in Fig. 3, and the thermodynamic binding parameters obtained are listed in Table 2. Titration of CaCl2into apoCML36-C at physiological
salt concentrations (Fig. 3A, black line) produced a multiphasic binding isotherm with an initial exothermic phase followed by an endothermic phase. The data best fit a four-site sequential binding model that reflected the presence of two classes of binding sites (Table 2): the first class comprises two
high-affin-Figure 3. ITC analysis of Ca2ⴙbinding to apoCML36-C and Mg2ⴙ-bound CML36-C (A) and Mg2ⴙbinding to apoCML36-C (B). Representative data curves
are shown of the calorimetric titrations of 4 mMCaCl2into apoCML36-C (A, black lines) and Mg2⫹-bound CML36-C (A, red lines) and of calorimetric titrations of
5 mMMgCl2into apoCML36-C (B). The upper panels represent raw traces minus baseline of the calorimetric titrations, and the lower panels show the
corre-sponding integrated binding isotherms. Under all applied conditions, the reference baseline data obtained by Mg2⫹and Ca2⫹ions titrated into buffer only
showed very weak dilution heat produced during the reaction (data not shown). The ligand dilution blank experiments were then subtracted from the binding isotherm obtained in the presence of protein.
Table 1
Determination of hydrodynamic radius of apo- and holoCML36-C by pulsed-field gradient diffusion NMR analysis and SEC
The mean values⫾ S.E. from triplicate experiments are presented. D, diffusion coefficient; Rh, hydrodynamic radius.
Pulsed-field gradient diffusion NMR
SEC Rh D Rh 10⫺10m2s⫺1 Å Å CML36-C⫹ EGTA 0.99 ⫾ 0.02 25.6⫾ 1.2 29.5⫾ 0.02 CML36-C⫹ Mg2⫹ 1.08⫾ 0.08 21.3⫾ 2.3 25.0⫾ 0.03 CML36-C⫹ Ca2⫹ 1.19⫾ 0.02 20.9⫾ 0.7 23.2⫾ 0.2 Table 2
Thermodynamics of Ca2ⴙbinding to CML36-C in the absence and
presence of Mg2ⴙ
The mean values⫾ S.E. from triplicate experiments using at least two different CML36-C preparations are presented.
No Mg2ⴙ ⴙ5 mMMg2ⴙ
Ka ⌬H Ka ⌬H
M⫺1 kcal mol⫺1 M⫺1 kcal mol⫺1
Site 1 1.1E7⫾ 0.6E7 ⫺11.9 ⫾ 0.5 3.5E6⫾ 0.5E6 ⫺6.6 ⫾ 0.1 Site 2 8.2E6⫾ 0.9E6 ⫺9.6 ⫾ 0.6 1.8E6⫾ 0.3E6 ⫺6.1 ⫾ 0.2 Site 3 9.6E3⫾ 0.8E3 3.6⫾ 0.4 8.7E3⫾ 1.1E3 2.8⫾ 0.1 Site 4 2.9E3⫾ 0.2E3 2.2⫾ 0.1 3.2E3⫾ 0.4E3 2.0⫾ 0.3
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ity sites (Kd1⫽ 91 ⫾ 17 nMand Kd2⫽ 122 ⫾ 13 nM, respectively)
with a heat release between⫺12 and ⫺10 kcal/mol of CaCl2;
and the second class likely represents the binding of two Ca2⫹
ions with lower affinity (Kd3⫽ 104 ⫾ 9Mand Kd4⫽ 345 ⫾ 24 M) with⌬H ⬃ 3 kcal/mol of CaCl2. The Ca2⫹titration was also
performed in the presence of 5 mMMg2⫹(Fig. 3A, red line, and
Table 2). Mg2⫹did not greatly affect the affinity constants asso-ciated with Ca2⫹binding to sites 3 and 4 of CML36-C, indicat-ing no bindindicat-ing of Mg2⫹to these sites, whereas the Kdvalues of sites 1 and 2 were shifted to lower affinity. Moreover, Mg2⫹had an effect on the⌬H of sites 1 and 2, as the process was less exothermic than that without Mg2⫹. This behavior, likely
sug-gesting that in the presence of Mg2⫹these two sites could be
formed and structurally stable, prompted us to quantitate the direct binding of Mg2⫹to CML36-C. Titration of MgCl
2into
apoCML36-C resulted in an exothermic binding isotherm (Fig. 3B), giving the best fit with the binding of two Mg2⫹ions to protein with a Kavalue of 9.2E3⫾ 0.7E3M⫺1(which corre-sponds to a Kdvalue of 109⫾ 8M), an enthalpy⌬H of ⫺3.9 ⫾ 0.7 kcal mol⫺1.
The stoichiometry of Ca2⫹ and Mg2⫹binding was further confirmed by analysis of the1H-15N HSQC NMR spectra of CML36-C. Downfield-shifted NMR peaks at⬃10.5 ppm are typical of conserved glycines at position 6 of the EF-hands occupied by divalent metals (25, 33, 34). In Ca2⫹-saturated
CML36-C, the presence of four downfield-shifted peaks corre-sponding to Gly-84 (EF-1), Gly-121 (EF-2), Gly-157 (EF-3), and Gly-194 (EF-4) confirms that Ca2⫹is bound to the four EF-hands, as seen by ITC analysis (Fig. 4A). The addition of satu-rating amounts of Mg2⫹to apoCML36-C caused the appear-ance of two downfield-shifted NMR peaks, assigned to Gly-157 and Gly-194 (Fig. 4B, green), indicating that Mg2⫹is bound to EF-3 and EF-4. Notably, the addition of Ca2⫹to Mg2⫹-bound
CML36-C caused the two downfield peaks at 10.55 ppm (Gly-157) and 10.45 ppm (Gly-194) to disappear and the appearance of the four glycine peaks in the position typical of the Ca2⫹
-bound protein (Fig. 4B, red), suggesting that the Ca2⫹ion could
displace Mg2⫹from EF-3 and EF-4.
CML36 interacts with ACA8 and stimulates its Ca2ⴙ-dependent
hydrolytic activity
To fully understand the roles of Ca2⫹sensors, the
identifica-tion of physiologically relevant targets is essential. A bioinfor-matics search for possible interactors of CML36 using string database (http://string-db.org/), which gives extensive cover-age and ease of access to both experimental and predicted inter-action information (35), found several promising candidates with various confidence scores. Among all of the putative pre-dicted interactors, we decided to focus on the PM-localized type IIB Ca2⫹-ATPase isoform ACA8 because: (i) it is a prom-inent in vivo regulator of cellular Ca2⫹ dynamics (18); (ii)
CML36transcript has been found to be altered in an
Arabidop-sisdouble mutant lacking both ACA8 and ACA10, another PM-localized Ca2⫹-ATPase isoform (36); and (iii) in a phosphopro-teomic study, CML36 has been shown to be associated with the PM fraction, which might facilitate the interaction with ACA8
in vivo(37). ACA8 possesses an extended cytosolic N-terminal regulatory domain with autoinhibitory function (16, 38 – 40). The best-known regulator of ACA8 is CaM. CaM binding to the N terminus induces a conformational change that suppresses autoinhibition, thus determining the activation of the pump. The N terminus is the target of modulation not only by CaM but also by other regulators of the pump, such as acidic phospho-lipids, which counteract its autoinhibitory action (41). In addi-tion, the phosphoregulation of N-terminal serine residues by influencing autoinhibition (42) is associated with alterations of the intracellular Ca2⫹ dynamics in response to mechanical
wounding (18). As a first step toward experimental validation of the putative interaction between CML36 and ACA8, we per-formed an in vitro overlay assay by spotting increasing amounts of recombinant CML36-C onto nitrocellulose and testing its ability to bind purified ACA8. We used bovine CaM and lysozyme as the positive and negative controls, respectively. Moreover, we tested another member of the Arabidopsis CML family, CML14 (43), which was not predicted to interact with ACA8. Specific interaction of ACA8 with CML36-C and CaM was evident (Fig. 5A). A signal of interaction with CML14 was
Figure 4. Zoom view of the1H-15N HSQC NMR spectra of the glycine downfield peaks of15N-CML36-C. A, portion of the HSQC spectrum of CML36-C in
complex with Ca2⫹ion. The assignment of the glycines of the EF-hands is indicated by the position number of the residue. B, superimposition of HSQC spectra
recorded on15N-CML36-C after the addition of Mg2⫹(green), Ca2⫹(black), and Ca2⫹into Mg2⫹-saturated protein (red). The spectra were recorded at 600 MHz
and 25 °C. All samples were at a protein concentration of 500Min 20 mMTris-HCl, 1 mMDTT, pH 7.5.
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visible, but a similar signal intensity was also observed when the nitrocellulose was incubated in the absence of ACA8 (Fig. 5A), strongly suggesting nonspecific binding of the ACA8 anti-body to CML14.
The specificity of the ACA8-CML36 interaction was also supported by native PAGE analysis (Fig. 5B). We produced in recombinant form the N-terminal portion of ACA8 corre-sponding to the regulatory domain (residues 1–116, named ACA8-N), and we checked the interaction between ACA8-N and both CML36-C and CML36 f.l. Recombinant Arabidopsis CaM1 (AtCaM1) was used as positive control and two other CMLs, i.e. CML14 and CML19, as negative controls. When ACA8-N and CML36 (both CML36-C and CML36 f.l. variants) were mixed in the presence of Ca2⫹, an additional band appeared on the native gel, likely representing the CML36-ACA8-N complex. An extra band was also visible for the AtCaM1-ACA8-N complex, whereas it was missing in the CML19- and CML14-containing samples, indicating the inabil-ity of these two CML isoforms to form a complex with ACA8-N. These results clearly confirmed the ability of CML36 to interact with ACA8 and localized the CML-binding domain to a region in the N terminus of the enzyme.
To elucidate the relevance of the CML36-ACA8 interaction, we subsequently examined its effect on ACA8 activity by mea-suring, in the presence of increasing concentrations of Ca2⫹ -bound CML36, the hydrolytic activity of ACA8 overexpressed in the yeast triple mutant K616, which is defective in high-affinity endogenous Ca2⫹pumps (38, 40, 44). Fig. 6A shows that the Ca2⫹-dependent hydrolytic activity of ACA8 was
signifi-cantly stimulated by CML36-C in a concentration-dependent manner. The concentration of CML36-C giving half-maximal stimulation of ACA8 activity (K0.5) was 534⫾ 42 nM. The
acti-vation of the Ca2⫹-ATPase was specific for CML36-C, as the addition of similar concentrations of two other CMLs, CML14 (Fig. 6A) and CML19 (data not shown), was completely ineffec-tive. To exclude the possibility that the CML36-C might lack portions with a potential regulatory role, we also performed the same experiment with CML36 f.l. The results showed that the
ACA8 activation curve in the presence of increasing concentra-tions of CML36 f.l. was comparable to that obtained using the truncated variant, CML36-C (supplemental Fig. S1).
The effect of CML36 on ACA8 activity was further evaluated in the presence of saturating CaM. As expected, the enzymatic activity of ACA8 was increased by CaM (42, 45). The addition of increasing concentrations of CML36-C did not influence CaM-stimulated enzymatic activity (Fig. 6A), revealing that the two effects were not additive. Thus, in accordance with the native PAGE results, we determined that CML36 and CaM interact within the N-terminal region of ACA8. Consistently, the response to CML36-C of the two N-terminal truncated mutants of ACA8 (⌬74ACA8and⌬109ACA8,fromwhichthehigh-affinityandboth the high- and low-affinity CaM-binding sites, respectively, have been deleted (38, 46)) was completely suppressed (Fig. 6B).
Because the above-described experiments were carried out on yeast microsomes overexpressing ACA8, to further corrob-orate our results we evaluated the response to CML36-C of ACA8 following a one-step purification on CaM-Sepharose. Fig. 6C shows that, under the experimental conditions used, the ACA8 activation curve resembled that obtained using the microsomal fraction, with an estimated K0.5value of 797⫾ 168
nM. Moreover, we confirmed the specificity of the CML36-C effect, as no ACA8 activation was detected in the presence of the other two CMLs, i.e. CML14 (Fig. 6C) and CML19 (data not shown). As shown in Fig. 6A, ACA8 was not further stimulated by CML36-C when activated by saturating CaM. We then tested the ability of 4.2MCML36-C to stimulate the ATPase
activity in the presence of increasing, but subsaturating, CaM concentrations. The results, reported in Fig. 6D, indicate an addi-tive effect of CML36-C and CaM that was evident only in the pres-ence of low CaM concentrations. This finding is in accordance with competition between the two activating proteins.
Expression pattern analysis of CML36 in different Arabidopsis organs
Quantitative RT-PCR performed on adult Arabidopsis plants indicated that the CML36 gene is ubiquitously expressed but is
Figure 5. ACA8/CML36-binding assays. A, overlay assay. Twol of native Ca2⫹-bound CML36-C corresponding to 0.6, 0.12, or 0.03 nmol was spotted onto 0.2
m nitrocellulose. After a cross-linking step, the overlay assay was performed by incubating the membrane without (upper panel) or with (lower panel) 1.5 M
purified ACA8 in the presence of 200MCaCl2and 2 mMMgSO4as described under “Experimental procedures.” Detection was performed using an anti-ACA8
antiserum. As a control, 0.6 nmol of CML14, 0.6 nmol of lysozyme, and 0.03 nmol of bovine testes CaM were used. Data are from one experiment representative of three that gave similar results. B, native PAGE analysis of CML36-C, CML36 f.l., AtCaM1, CML14, and CML19 in the absence and presence of ACA8-N in 100 mM
Tris-HCl, 5 mMCaCl2, and 4Murea, pH 7.5.
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more abundant in roots and inflorescences (Fig. 7A and
sup-plemental Fig. S2A). This expression pattern showed a
sim-ilar trend when either actin (Fig. 7A) or a subunit of phos-phatase 2A (47) (supplemental Fig. S2A) was used as a reference gene for normalization. Our data agree with the
Arabidopsismicroarray expression data available at AtGen-Express Visualization Tool. ACA8 expression was found in all organs examined, with the highest levels in inflorescences (Fig. 7B andsupplemental Fig. S2B) (48).
Discussion
Given the abundance of CaM/CML in plants, the definition of their properties as Ca2⫹ sensors and the identification of their downstream targets represent an impressive challenge. Herein, we have presented structural and biochemical studies demonstrating that recombinant CML36 from A. thaliana dis-plays the typical properties of proteins that function as Ca2⫹
sensors and can stimulate the hydrolytic activity of the
Arabi-dopsisACA8 isoform by specifically binding to the N-terminal regulative domain of ACA8. CML36 possesses the characteris-tics of regulatory EF-hand Ca2⫹-binding proteins including, upon Ca2⫹binding: (i) an increase in␣-helical content; (ii) a
shift in electrophoretic mobility; (iii) the adoption of a stable tertiary structure; and (iv) the exposure of a hydrophobic pocket that can interact with an ANS probe as a result of the Ca2⫹-induced conformational changes, which is often a critical
process for the function of many Ca2⫹sensor proteins. In
addi-tion, we obtained information on the site-specific Ca2⫹binding
affinities of CML36, which are crucial because Ca2⫹affinity is often attuned to the physiological role of the Ca2⫹sensor
pro-tein. We found that CML36 possesses two mixed Ca2⫹/Mg2⫹ -binding sites of high affinity (KdCa⬍ 0.2Mand KdMg⬃ 100
M) and two Ca2⫹-specific sites of low affinity (ⱖ100M). The
NMR data indicate that CML36 binds the two Mg2⫹ ions to EF-3 and EF-4; therefore, EF-3 and EF-4 represent the Ca2⫹/
Mg2⫹high-affinity sites, whereas EF-1 and EF-2 are the
low-affinity sites. Notably, all four EF-hands of the protein are capa-ble of sensing Ca2⫹ions. This finding was unexpected, as only
three EF-hands were predicted to be functional Ca2⫹-binding sites by PROSITE-ProRule annotation (49). Indeed, EF-2 (resi-dues 108 –127) possesses an arginine residue at position 9 of the loop, where a hydrophilic or negative side chain is usually hydrogen-bonded to a Ca2⫹-ligand water molecule, and thus it
is not predicted to have functional Ca2⫹binding, although the
canonical Ca2⫹-coordinating groups at positions 1, 3, 5, 7, and 12 are present. It is tempting to attribute this EF-hand to the lowest-affinity site. Further, the reduced affinity of EF-1 could be ascribed to the replacement of a conserved glutamate resi-due with an aspartate at position 12 (8). Future studies of Ca2⫹
-bound CML36 X-ray structures will be helpful in elucidating the difference in the microenvironment of the EF-hands at the
Figure 6. Activation of ACA8 as a function of increasing CML36-C concentrations. Ca2⫹-ATPase activity of yeast microsomes overexpressing ACA8 (A) or
of the purified pump (C) was measured at the specified CML36-C concentrations in the presence (squares) or absence (circles) of 1MCaM. Activation by CML14
was also tested under the same experimental conditions (triangles). Enzyme stimulation (f/fmax) is expressed as the ratio between percent of activation at the indicated CML concentrations (f) and maximal activation evaluated at the highest CML concentration tested (fmax; 180 –350% in different biological samples).
B, basal (light gray bars) and CML36-C-stimulated (dark gray bars) hydrolytic activity of ACA8 full-length (FL),⌬74ACA8, and ⌬109ACA8. Ca2⫹-dependent ATPase
activity was assayed in yeast microsomes expressing different versions of ACA8 in the presence of 1.9MCML36-C. Activities⫾ S.E. were normalized, setting
as 100 the activity measured in the presence of 3MCaM to compensate for different levels in protein expression. CaM-stimulated activity ranged between 70
and 170 nmol Pimin⫺1mg⫺1protein in different biological samples. D, Ca
2⫹-ATPase activity of purified ACA8 measured at the indicated CaM concentrations
with (gray bars) or without (white bars) 4.2MCML36-C. Maximal activation (fmax) was evaluated at the highest CaM concentration tested.
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atomic level, which will facilitate understanding of these site-specific Ca2⫹affinity properties.
Notably, Mg2⫹binding to high-affinity EF-3 and EF-4 sites also induces a global conformational change throughout CML36 that results in the stabilization of its tertiary structure. Cation binding to the Ca2⫹/Mg2⫹mixed sites seems to control
an important structural transition from a molten globule state to a compact globular conformation. Indeed, we found that, similar to other Ca2⫹-binding proteins (24, 25, 28, 50),
apoCML36 likely assumes a molten globule structure and the transition from this molten globule to the compact state clearly occurs when the high-affinity sites are occupied by either Ca2⫹
or Mg2⫹.
The observed binding constants of Ca2⫹/Mg2⫹mixed sites
for Mg2⫹ and for Ca2⫹ imply that under resting conditions
these two EF-hands are always occupied by a divalent cation, ensuring a folded ion-bound structure of CML36 at any cyto-plasmic Ca2⫹concentration. Next, in response to the rapid increase in intracellular Ca2⫹concentrations upon a stimulus, the Ca2⫹-bound state becomes the dominant state of the
pro-tein, able to displace Mg2⫹from these EF-hands.
Beyond the description of the structural and metal-binding properties of CML36, a significant advancement in the knowl-edge about this protein has been obtained by the identification of ACA8 as a CML36 downstream target. As a strategical iso-form of PM-localized Ca2⫹pumps in the shaping of intracellu-lar Ca2⫹ dynamics (18), ACA8 is the target of regulation by multiple Ca2⫹-dependent effectors (17, 18, 42). We have
pro-vided direct evidence that ACA8 is also able to interact with the Ca2⫹sensor CML36 in vitro and that this interaction results in
the activation of its Ca2⫹-dependent hydrolytic activity with a K0.5value in the submicromolar range. Neither this interaction
nor the activation of ACA8 is detectable with other isoforms of CMLs such as CML19 and CML14, suggesting that ACA8 may be specific for CML36. Moreover, native PAGE analysis allowed us to identify the binding site for CML36 at the N-ter-minal regulative domain of ACA8. The interaction of CML36 with the N terminus of ACA8 is also supported by our observa-tions that the effects of CML36 and CaM on ACA8 activity are not additive and that variants of ACA8 without the N-terminal CaM-binding domains are completely insensitive to CML36. Nevertheless, further investigations will be necessary to
pre-cisely localize the CML-binding site within the N-terminal portion.
The capacity of both CaM and CML36 to bind ACA8 raises the question of which mechanisms are capable of avoiding pro-miscuous interactions in cell-regulatory processes. The choice of the proper signaling pathway involving CML36 or CaM may be related to many modulating factors: (i) the abundance of each protein in a specific microdomain, cellular compartment, tissue and organ; (ii) the time-dependent expression in differ-entiation and development; (iii) the temporal and spatial fea-tures of the Ca2⫹signal; (iv) the properties of the EF-hand pro-teins, such as the Ca2⫹ affinity, the number of functional
binding sites, and the ability to discriminate between Ca2⫹and Mg2⫹; and (v) site-specific phosphorylation. In this scenario, it
is worth noting that the predicted affinities of CML36 for Ca2⫹
differ significantly from those observed for mammalian and
ArabidopsisCaM1 (29, 51); this would influence the ability of these Ca2⫹ sensors to respond to cytosolic Ca2⫹increases induced by the perception of different stimuli and thus their ability to activate their downstream targets as ACA8. More-over, the CML36 and ACA8 genes are co-expressed mainly in shoot inflorescences. Furthermore, CML36 displays an elon-gated N terminus, which may represent a targeting sequence. Indeed, a mass spectrometry– based quantitative phosphopro-teomics approach identifies CML36 as a protein associated with the PM fraction (37), which might facilitate the interaction with ACA8 in vivo.
PM-localized Ca2⫹pumps are likely to play a key role in
returning cytosolic Ca2⫹to resting concentrations and in mod-ulating the dynamics of a Ca2⫹ signature (2, 5). Analysis of ACA8expression, which is up-regulated upon cold stress and abscisic acid treatment, supports its involvement in crucial pro-cesses such as response to abiotic stresses and hormonal regu-lation (48, 52). Furthermore, genetic evidence using KO plant mutants also indicates additional roles for ACA8 in develop-mental processes and plant immune response (36). Expression of the CML36 gene also suggests a possible role in organ devel-opment for CML36. Interestingly, the CML36 transcript has been found to be altered in an Arabidopsis double mutant lack-ing both ACA8 and ACA10, another PM-localized Ca2⫹ -ATPase isoform (36). In addition, ACA8 has been recently identified as a prominent in vivo regulator of cellular Ca2⫹
Figure 7. Analysis of CML36 and ACA8 expression in Arabidopsis. Quantitative RT-PCR of CML36 (A) and ACA8 (B) was performed in various organs of wild-type adult plants. The expression levels were normalized using actin as the endogenous control gene, and the relative expression ratios were calculated
using leaves as the calibrator sample. The values reported are means⫾ S.E. (n ⫽ 3); Student’s t test was applied: **, p ⬍ 0.01, and ***, p ⬍ 0.001 versus leaves.
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dynamics which crucially functions in the termination of Ca2⫹
signals induced by mechanical wounding and related stimuli (18). In this scenario, CML36 would act in concert with other Ca2⫹sensors such as CaM (16, 39, 53, 54), CDPK (42), and the CBL-CIPK complexes (18) to regulate ACA8 activity. In addi-tion, other CMLs may be involved in the modulation of ACA8 and other different isoforms of autoinhibited Ca2⫹ pumps, which might contribute to the elaborated/intricate Ca2⫹ -de-pendent regulatory network for the fine-tuning of intracellular Ca2⫹homeostasis.
Experimental procedures Protein production
The coding sequence for CML36 (At3g10190) was PCR-am-plified using pUNI51-U23411 vector as template obtained from The Arabidopsis Information Resource (TAIR). The following primers were employed: forward, 5⬘-CATATGCACCAC CACCACCACCACATGAAACTCGCCAAAC-3⬘; and reverse, 5 ⬘-GAATTCTCAACGCTGGAGATCCATCATTCGTGAG-3⬘. The forward primer carried a NdeI restriction site and a His6
tag, and the reverse primer contained a stop codon and an EcoRI restriction site. The verified cDNA sequence was then cloned into the corresponding restriction sites of pET21a vec-tor for expression in Escherichia coli BL21(DE3) cells. Cells were grown in LB medium at 37 °C to A600 nm⫽ 0.6, and
ex-pression of CML36 protein was then induced at 25 °C for 16 h by the addition of 0.4 mMIPTG. To express15N-labeled CML36
for NMR studies, M9 minimal medium supplemented with
15NH4Cl (1 g liter⫺1) was employed. The bacterial pellet
ob-tained after low-speed centrifugation of a 1-liter culture was solubilized in 20 mMTris-HCl, pH 7.5, 150 mMKCl, 10 mM
imidazole, and 1 mMDTT lysis buffer containing 0.1 mg ml⫺1
lysozyme, stirred at room temperature for 30 min, and then sonicated on ice. The lysate was centrifuged at 30,000⫻ g for 10 min at 4 °C, and the supernatant was loaded onto a nickel-affinity column pre-equilibrated in 20 mMTris-HCl, pH 7.5,
150 mMKCl, 1 mMDTT, and 10 mMimidazole buffer. A
gradi-ent of imidazole from 10 to 500 mMwas applied to the column, and the His-tagged CML36 was eluted around 100 mM
imidaz-ole. The fractions containing CML36 were pooled and washed extensively at 4 °C with 20 mMTris-HCl, pH 7.5, 150 mMKCl,
and 1 mMDTT using Vivaspin concentrators (Sartorius, Goet-tingen, Germany) to remove the imidazole. Protein concentra-tion was determined using the Bio-Rad assay.
cDNA encoding N-terminally truncated CML36 was gener-ated by PCR using the complete cDNA of CML36 as template, a forward primer containing a NdeI site (5 ⬘-CATATGCATCAT-CATCATCATCACGAGGTTCCTTCTCCTTATTC-3⬘) and a reverse primer containing a EcoRI site (5 ⬘-GAATTCT-CAACGCTGGAGATCCATCATTCGTGAG-3⬘). The result-ing DNA fragment was inserted into pET21a usresult-ing the NdeI/ EcoRI sites for directional cloning. The newly generated plasmid was checked by DNA sequencing and used to trans-form E. coli BL21(DE3) cells. The conditions for expression and purification of the deletion mutant were as described for CML36 f.l. The yield was about 6 mg and 100 mg for a 1-liter culture for the full-length protein and the N-terminally
trun-cated variant, respectively. Purified CML36 variants were treated with 2 mMEGTA, washed extensively against
decalci-fied buffer and then against decalcidecalci-fied NH4HCO3 buffer,
lyophilized, and stored at ⫺80 °C until use. Buffers were decalcified by using Chelex-100 ion-exchange resin (Sigma) according to the manufacturer’s instructions. The residual Ca2⫹ concentration was determined using 5,5⬘-Br2-BAPTA
(5,5 ⬘-dibromobis-(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tet-raacetic acid) as described (55, 56).
AtCaM1 and CML14 were produced as described (43, 57). CML19 was expressed and purified as reported in the
supple-mental “Methods”. The production of recombinant ACA8-N,
the N-terminal domain of ACA8 (residues 1–116) was per-formed as described (54, 58).
The Ca2⫹-induced mobility shift of CML36-C was
per-formed on a 12.5% Tris-glycine native PAGE and a 15% SDS-PAGE after incubating CML36-C with 5 mMCaCl2or 5 mM
EGTA for 30 min at room temperature.
Nuclear magnetic resonance spectroscopy
NMR spectra were collected on a 600-MHz Bruker Avance III spectrometer (Bruker, Karlsruhe, Germany) equipped with a cryogenic probe. A standard1H-15N HSQC pulse sequence was
used with pulsed-field gradients for suppression of the solvent signal.1H-15N HSQC experiments were collected with a data
matrix of 12 ppm (1H)⫻ 36 ppm (15N), 8 scans, and a 1.2-s
relaxation delay.
To achieve partial backbone atom assignment of CML36-C bound to Ca2⫹or Mg2⫹ions, standard triple resonance NMR experiments were acquired on 15N,13C-labeled samples (59).
The PFG-STE (pulsed-field gradient-stimulated echo) experi-ments, employing bipolar gradients (60), were conducted and analyzed as described previously (61). DSS (1 mMfinal
concen-tration) was added to the protein samples and used as an inter-nal standard (62). Delays of 25 and 180 ms between the defo-cusing and refodefo-cusing gradient elements were employed for experiments on DSS and protein samples, respectively. Spectra were acquired with 8000 complex points and 128 transients. The Rhvalues of the proteins were calculated based on the rela-tionship Rb
prot⫽ (D
DSS/Dprot) Rb
ref, where R
b
refis the
hydrody-namic radius reported for DSS (3.43 Å) and DDSSand Dprotare the diffusion coefficients calculated as described for the DSS and the protein, respectively. The spectra were recorded at 25 °C in 20 mMTris-HCl, 1 mMDTT, pH 7.5, at a protein
con-centration of 500Min the presence of 5 mMEGTA, 5 mM
MgCl2, or 5 mMCaCl2and analyzed with Topspin3.2 (Bruker,
Karlsruhe, Germany).
Size-exclusion chromatography
The hydrodynamic radius of apoCML36-C and Mg2⫹- and Ca2⫹-bound CML36-C was estimated by SEC using a Superose 12 column (10/300GL, GE Healthcare) as described previously (43, 63). Each experiment was performed at least in triplicate, and the reported values represent means⫾ S.E.
Spectroscopic measurements
CD spectra were collected on a Jasco J-710 spectropolarime-ter at 25 °C in 20 mMTris-HCl,150 mMKCl, and 1 mMDTT, pH
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7.5, in the presence of 5 mMEGTA, 5 mMCaCl2,or 5 mMMgCl2.
Protein concentration was ⬃90Min a cuvette with a path
length of 1 cm for near-UV wavelengths, and for far-UV spectra the CML36-C concentration was 10 M in a cuvette with a
0.1-cm path length. Three spectra, averaged automatically, were recorded for each sample as described (29). The ANS fluo-rescence spectra were recorded on a Jasco FP8200 spectrofluo-rometer as described (29).
Isothermal titration calorimetry
ITC experiments were performed on a TA Instruments Nano ITC as described (29, 43). All solutions were degassed prior to the rinsing and loading of both the titration and sample cells. The buffer used was decalcified 20 mMTris-HCl, 150 mMKCl,
pH 7.5. Lyophilized CML36-C was resuspended in decalcified buffer.
For Ca2⫹titrations into 200MapoCML36-C or Mg2⫹
-sat-urated CML36-C, 4 mMCaCl2was injected with a 0.3-l
injec-tion to displace air from the syringe followed by 40⫻ 1.25-l injections. Mg2⫹-saturated CML36-C was prepared by
incu-bating apoCML36-C for 15 min at 25 °C with 5 mMMgCl2.
For experiments in which Mg2⫹ was injected into the
apoCML36-C, a 5 mM MgCl2 stock was added to 170 M
apoCML36 by a 1-l injection to displace air from the syringe followed by 24⫻ 2-l injections.
The heats of dilution, collected by injecting Ca2⫹or Mg2⫹ into buffer without the protein in the sample cell, were sub-tracted from the raw integrated heats in the presence of protein. Data were fitted using the best-fitting model for each experi-ment (sequential, two-site, or one-site model). The reported values represent the mean⫾ S.E. of at least three independent titrations.
The fitted Kavalues were converted to Kdvalues using the equation,
Kd⫽ 1
Ka
(Eq. 1)
Native PAGE analysis
The formation of the complex following ACA8 binding was analyzed under native conditions as described previously (29). Briefly, CML36 f.l., CML36-C, Arabidopsis CaM1, CML14, and CML19 were dissolved at a 10Mconcentration in 100 mM
Tris, pH 7.5, 5 mMCaCl2, and 4Murea and incubated in the
presence of an excess of recombinant ACA8-N for 1 h at room temperature. Samples were assayed on a 12.5% continuous gel containing 5 mMCaCl2and 4 M urea. Urea was added
either to the gel and the incubation buffer to prevent any nonspecific interactions.
Yeast strain and isolation of microsomes overexpressing ACA8
Saccharomyces cerevisiaestrain K616 (MAT␣ pmr1:: HIS3 pmc1:: TRP1 cnb1::LEU2, ade2, ura3; (44)) was used for ACA8 expression (38). Microsomes from yeast cells expressing full-length ACA8 and ACA8-deleted mutants (40, 46) were pre-pared as reported previously (38). Protein concentration was determined using the Bio-Rad assay.
ACA8 purification from microsomes
Yeast microsomes were solubilized as reported (45) and incubated (45 mg of microsomes) overnight at 4 °C on 4 ml of CaM-Sepharose 4B gel (GE Healthcare). Purification was per-formed as described (45). The 1 mM EGTA-eluted fraction,
added with stoichiometric CaCl2, was concentrated about
70-fold on Vivaspin ultrafiltration spin columns, with a 30-kDa cutoff (Sartorius, Goettingen, Germany), and immediately used for testing Ca2⫹-ATPase activity or for overlay analysis. For quantification, 1l of the concentrated eluate was loaded onto a precast Tris-Tricine polyacrylamide gel (4 –12% linear gradi-ent; Anamed, Darmstadt, Germany) with increasing amounts of -galactosidase used as the standard. After staining with Coomassie Blue, signal quantification was carried out using AlphaEaseFC software by Alpha-Innotech (MMedical, MI, Italy). ACA8 was purified to virtual homogeneity.
Ca2ⴙ-ATPase activity assay
ACA8 activity in yeast microsomes (0.4 mg of protein/ml) and in the EGTA-purified fraction (0.03 mg of protein/ml) was assayed as Ca2⫹-dependent MgITP or MgATP hydrolysis,
respectively, as described (40, 42). Free Ca2⫹concentration was buffered at 10Mwith 1 mMEGTA. Bovine testes CaM
(Sigma-Aldrich) and A. thaliana purified Ca2⫹-bound CML36 or CML14 were added at the specified concentrations. Assays were performed by preincubating CMLs with microsomes or ACA8-purified protein in assay buffer for 20 min at 25 °C before starting the reaction by the addition of nucleotide sub-strates. Samples were then incubated at 25 °C for 60 min. MgITP or MgATP hydrolysis measured in the presence of 1 mM
EGTA without added Ca2⫹was subtracted from the reported
data. Assays were performed at least four times, with three rep-licates for each condition, using three independent biological samples and at least two different CML36 preparations. The standard error did not exceed 5% of the mean. Data analysis and curve fitting were performed using SigmaPlot software. K0.5 for CML36 (⫾S.E.) was estimated using nonlinear regression. Student’s t test was conducted to assess potential differences between K0.5estimated from data obtained using yeast micro-somes and purified ACA8 (p⫽ 0.1173).
Overlay assay
Different amounts of purified Ca2⫹-bound CML36 (0.6, 0.12, and 0.03 nmol), 0.6 nmol of purified Ca2⫹-bound CML14, 0.6
nmol of lysozyme (Sigma-Aldrich), and 0.03 nmol of bovine testes CaM (Sigma-Aldrich) were spotted onto 0.2-m nitro-cellulose. Proteins were first cross-linked to the membrane by incubating them for 45 min at room temperature in 0.2% (v/v) glutaraldehyde freshly prepared in KP buffer (25 mM KH2/
K2HPO4buffer, pH 7). The membrane was incubated
thereaf-ter for 16 h in blocking solution (3% (w/v) BSA, 0.15MNaCl, 2 mMMgSO4, 0.2% (v/v) polyoxyethylene (20) sorbitan
monolau-rate, and 20 mMTris-HCl, pH 7.4). After washing, the overlay
was conducted by incubating the membrane for 2 h at room temperature in blocking solution with 1.5Mpurified ACA8 or,
in parallel as a negative control, in the same solution without ACA8. Following a fast wash in blocking solution, an additional 45-min cross-linking step was carried out. Immunodetection
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with an antiserum against the sequence268
EISIYDIVVGDVIP- LNIGNQVPADGVLISGHSLALDESSMTGESKIVNKDANK-DPFLMSGCKVADGNGSMLVTGVGVNTEW348 of ACA8 was performed as described (58).
Plant material, growth conditions, and gene expression analysis by quantitative reverse transcription-PCR
To evaluate CML36 gene expression in various adult organs,
A. thalianaecotype Columbia (Col-0) seeds were sown on soil in a greenhouse under a 16-h light/8-h dark photoperiod at 24 °C and 20 °C, respectively; the plants were grown to matu-rity. The isolation of total RNA was performed using the RNeasy mini kit (Qiagen, Hilden, Germany) from 100 mg of frozen organs, and then the RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI). cDNA syn-thesis was obtained with oligo-(dT) primer and carried out using the GoScript reverse transcription system (Promega). SYBR Green qPCR Supermix-UDG (Invitrogen) was used to amplify cDNA on the StepOnePlusTMreal-time PCR system (Applied Biosystems, Foster City, CA). Quantitative RT-PCR was performed using the following cycling conditions: 2 min at 50 °C, 2 min at 95 °C, 40 cycles of 95 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and finally 72 °C for 3 min. The actin gene was used as an endogenous control and to normalize all quantifica-tions. Three cDNA samples obtained from three independent RNA extractions were examined. Relative quantitation of tran-script levels was performed as reported (64). The following forward (F) and reverse (R) primers were employed: for
CML36(At3g10190), F, 5⬘-CATCTTCCGGCCAAGACTGT-3⬘, and R, 5⬘-ACCTCCGGGAGAATGCTAGT-3⬘; for ACA8 (At5g57110), F, 5⬘-AAGTCCTTACCGTAGCGGTTACAA-3⬘ and R, 5⬘-ATTGAATAGGCAAGGGTTAAAGTAACA-3⬘ (48). Two internal reference genes, an actin gene (At3g18780) and a subunit of phosphatase 2A (PP2A At1g13320), were used for data normalization (47, 65). The following primers were employed: for actin, F, 5 ⬘-TGTTCTCTCCTTGTACGC-CAGT-3⬘, and R, 5⬘-CAGCAAGGTCAAGACGGAGGA-3⬘; and for PP2A, F 5⬘-TAACGTGGCCAAAATGATGC-3⬘, and R, 5⬘-GTTCTCCACAACCGCTTGGT-3⬘ (47).
Author contributions—A. A. and P. D. conceived the study. A. A. coordinated the study and wrote the paper. A. A., R. V., and V. L. V. produced the recombinant proteins, designed, performed, and ana-lyzed the structural, metal-binding, and native PAGE experiments. M. C. B. and L. L. carried out overlay and activity assays. M. C. B. participated in drafting the article and revising it critically. M. D. performed the NMR experiments. B. M. carried out gene expression analysis. All authors analyzed the results and approved the final ver-sion of the manuscript.
Acknowledgments—We are grateful to Prof. M. I. De Michelis and Prof. A. Costa (University of Milano) for fruitful discussions and crit-ical reading of the manuscript. We thank Prof. M. Bertoldi (University of Verona) for help with the CD experiments. We also thank the “Cen-tro Piattaforme Tecnologiche” of the University of Verona for provid-ing access to the NMR spectrometer and nanoITC calorimeter.
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